The rhizobacterium Serratia plymuthica 4Rx13 releases a unique polymethylated hydrocarbon (CH) with a bicyclo[3.2.1]octadiene skeleton called sodorifen. Sodorifen production depends on a gene cluster carrying a C-methyltransferase and a terpene cyclase along with two enzymes of the 2- C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis. Comparative analysis of wild-type and mutant volatile organic compound profiles revealed a C-methyltransferase-dependent C alcohol called pre-sodorifen, the production of which is upregulated in the terpene cyclase mutant. The monocyclic structure of this putative intermediate in sodorifen biosynthesis was identified by NMR spectroscopy. In vitro assays with the heterologously expressed S. plymuthica C-methyltransferase and terpene cyclase demonstrated that these enzymes act sequentially to convert farnesyl pyrophosphate (FPP) into sodorifen via a pre-sodorifen pyrophosphate intermediate, indicating that the S-adenosyl methionine (SAM)-dependent C-methyltransferase from S. plymuthica exhibits unprecedented cyclase activity. In vivo incorporation experiments with C-labeled succinate, l-alanine, and l-methionine confirmed a MEP pathway to FPP via the canonical glyceraldehyde-3-phosphate and pyruvate, as well as its SAM-dependent methylation in pre-sodorifen and sodorifen biosynthesis.C{H} NMR spectroscopy facilitated the localization of C labels and provided detailed insights into the biosynthetic pathway from FPP via pre-sodorifen pyrophosphateto sodorifen.
Many and complex plant-bacteria inter-relationships are found in the rhizosphere, since plants release a variety of photosynthetic exudates from their roots and rhizobacteria produce multifaceted specialized compounds including rich mixtures of volatiles, e.g., the bouquet of Serratia odorifera 4Rx13 is composed of up to 100 volatile organic and inorganic compounds. Here we show that when growing on peptone-rich nutrient medium S. odorifera 4Rx13 and six other rhizobacteria emit high levels of ammonia, which during co-cultivation in compartmented Petri dishes caused alkalization of the neighboring plant medium and subsequently reduced the growth of A. thaliana. It is argued that in nature high-protein resource degradations (carcasses, whey, manure and compost) are also accompanied by bacterial ammonia emission which alters the pH of the rhizosphere and thereby influences organismal diversity and plant-microbe interactions. Consequently, bacterial ammonia emission may be more relevant for plant colonization and growth development than previously thought.
Bacteria emit a wealth of volatile organic compounds. Gas chromatography coupled to mass spectrometry analysis of five Serratia strains revealed ketones, dimethyl di- and trisulfide and 2-phenylethanol commonly released in this genus. The polymethylated bicyclic hydrocarbon sodorifen was uniquely released by the rhizobacterium Serratia plymuthica 4Rx13. Of 10 Serratia strains, only S. plymuthica isolates originating from plants grown on fields near Rostock (Germany) released this new and unusual compound. Since the biosynthetic pathway of sodorifen was unknown, the genome sequence of S. plymuthica 4Rx13 was determined and annotated. Genome comparison of S. plymuthica 4Rx13 with sodorifen non-producing Serratia species highlighted 246 unique candidate open reading frames.
Bacteria release a plethora of volatile organic compounds, including compounds with extraordinary structures. Sodorifen (IUPAC name: 1,2,4,5,6,7,8-heptamethyl-3-methylenebicyclo[3.2.1]oct-6-ene) is a recently identified and unusual volatile hydrocarbon that is emitted by the rhizobacterium Serratia plymuthica 4R×13. Sodorifen comprises a bicyclic ring structure solely consisting of carbon and hydrogen atoms, where every carbon atom of the skeleton is substituted with either a methyl or a methylene group. This unusual feature of sodorifen made a prediction of its biosynthetic origin very difficult and so far its biosynthesis is unknown. To unravel the biosynthetic pathway we performed genome and transcriptome analyses to identify candidate genes. One knockout mutant (SOD_c20750) showed the desired negative sodorifen phenotype. Here it was shown for the first time that this gene is indispensable for the synthesis of sodorifen and strongly supports the hypothesis that sodorifen descends from the terpene metabolism. SOD_c20750 is the first bacterial terpene cyclase isolated from Serratia spp. and Enterobacteriales. Homology modeling revealed a 3D structure, which exhibits a functional role of amino acids for intermediate cation stabilization (W325) and putative proton acception (Y332). Moreover, the size and hydrophobicity of the active site strongly indicates that indeed the enzyme may catalyze the unusual compound sodorifen.
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